This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Improved understanding of the structural basis of electrostatic effects in proteins is necessary for correlation of structure with function. Most previous studies have focused on surface ionizable residues. Their general properties and their contributions to stability and function are relatively well understood. One of the remaining problems in protein electrostatics concerns the properties of internal ionizable groups. The pKa values of these groups are governed by dielectric properties of proteins that are poorly understood. To examine this issue we engineered a family of proteins with ionizable groups (Lys, Arg, Asp, Glu) at 25 internal positions. A hyperstable variant of staphylococcal nuclease was used for these purposes. 98 out of 100 variants that were made fold into a native-like state. The pKa values of these internal residues have been measured. The majority of the pKa values are shifted in the direction that promotes the neutral state (elevated for acidics and depressed for basic residues), some by nearly 6 pKa units. These shifts suggest that the dehydration experienced by the ionizable groups in their buried positions is not fully compensated for by contacts with polar or ionizable groups. The structures are useful because they contain information about factors that contribute towards the pKa values of the internal ionizable residues. The complex response of the protein to the ionization of an internal group is likely to involve conformation reorganization or changes in dynamics. pKa values of internal ionizable groups serve as a benchmark to test different computational methods. Continuum electrostatic calculations fail to self consistently reproduce these experimentally measured pKa values of internal ionizable group. This point to the lack of understanding of the nature of protein dielectric response. We are investigating the details of microenvironments of internal ionizable groups and correlating them to their pKa shifts. Understanding pKa determinants of internal ionizable residues has great implication for binding studies in drug design, rational protein engineering work, and any computational work that uses protein structure as a starting point.